2.3 | Miniaturization and intracellular accumulation of droplets
Next, the lipid-coated PFH droplets were miniaturized and examined for uptake into living cells through endocytosis. The diameter of endosomes has been reported to be less than 120 nm[5], and we wanted the droplets to be smaller than this. Therefore, the droplets were extruded through 30 nm pore filters, and their average diameter was determined to be 66.8 nm using dynamic light scattering (Figure 2B). The present PCNDs were modified with biotinylated and fluorescently labeled 9E5 through cross-linking with NeutrAvidin. Alexa Fluor 647 (AF647) was used for labeling 9E5. In addition, a fluorescently labeled PEG-lipid (tetramethyl rhodamine (TAMRA)-labeled PEG-lipid, DPPE-PEG-TAMRA) (Figure 1; also see Supporting Information, Figure S1) was incorporated into the lipid coating of the droplets to visualize the PCNDs. In this study, human colon carcinoma DLD1 cells were used as the model EREG-expressing cancer cells. After the treatment of DLD1 cells with 9E5-conjugated PCNDs for 2 h at 37 ᵒC, the fluorescence of both TAMRA and AF647 was observed using confocal fluorescence microscopy at almost the same location inside the cells (Figure 2C). In the case of non-conjugated PCNDs, no fluorescence was observed inside the cells (Figure 2D). These results indicate that the present PCNDs were taken up into living cells due to 9E5 conjugation, as previously reported[30,31].